This proposal will investigate, at the molecular level, changes in chromatin structure and genetic activity of mammalian gametes and, eventually, the early embryo. Initial studies will focus on examining the chromatin organization of the mature mouse sperm nucleus before fertilization. Biochemical methods will be used for analyzing the subunit and higher order structure of sperm chromatin and the correlation of methylation of cytosine residues with gene expression of the male germ cell. The specific objectives of the proposed experiments are: (1) to define the basis unit structure in chromatin of the mature mouse spermatozoon, which contains protamine-like proteins, compared to the nucleosomal-type of organization found in the histone-containing chromatin of somatic cells and pachytene spermatocyes; (2) to elucidate the functional role of protamine-like proteins in the organization of the mature sperm nucleus by comparing the structures of transcriptionally active and inactive DNA sequences in earlier spermatogenic stages versus mature sperm; and (3) to examine changes in the pattern of distribution of methyl-cytosine residues in specific regions of the mouse genome at various stages of development and differentiation of germ-line cells. Experimental approaches for each of the specific objectives are similar and involve primarily filter hybridization (Southern blot) analysis of DNA obtained from isolated nuclei of individual cell types treated with nucleases under a variety of conditions. The results obtained from these experiments will provide important new information on the basic organization of DNA and proteins in the nuclei of mammalian cells and on the function of this organization in regulating gene expression in germ cells and early embryos.